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Nucleic Acids Research 2004 32(14):4377-4389; doi:10.1093/nar/gkh772
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Published online 16 August 2004

Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved

SELEX-derived aptamers of the duck hepatitis B virus RNA encapsidation signal distinguish critical and non-critical residues for productive initiation of reverse transcription

Kanghong Hu, Jürgen Beck and Michael Nassal*

University Hospital Freiburg, Department of Internal Medicine II/Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany

* To whom correspondence should be addressed. Tel: +49 761 270 3507; Fax: +49 761 270 3507; Email: nassal2{at}ukl.uni-freiburg.de
Present address: Kanghong Hu, University Hospital Regensburg, Department of Internal Medicine I, Forschungsbau H1, R38, D-93042 Regensburg, Germany

Received July 22, 2004; Accepted July 28, 2004

Protein-primed replication of hepatitis B viruses (HBVs) is initiated by the chaperone dependent binding of the reverse transcriptase (P protein) to the bulged {epsilon} stem-loop on the pregenomic RNA, and the {epsilon}-templated synthesis of the 5' terminal nucleotides of the first DNA strand. How P protein recognizes the initiation site is poorly understood. In mammalian HBVs and in duck HBV (DHBV) the entire stem-loop is extensively base paired; in other avian HBVs the upper stem regions have a low base pairing potential. Initiation can be reconstituted with in vitro translated DHBV, but not HBV, P protein and DHBV {epsilon} (D{epsilon}) RNA. Employing the SELEX method on a constrained library of D{epsilon} upper stem variants, we obtained a series of well-binding aptamers. Most contained C-rich consensus motifs with very low base pairing potential; some supported initiation, others did not. Consensus-based secondary mutants allowed to pin down this functional difference to the residues flanking the conserved loop, and an unpaired U. In vitro active consensus sequences also supported virus replication. Hence, most of the upper stem acts as a spacer, which, if not base paired, warrants accessibility of relevant anchor residues. This suggests that the base paired D{epsilon} represents an exceptional rather than a prototypic avian HBV {epsilon} signal, and it offers an explanation as to why attempts to in vitro reconstitute initiation with human HBV have thus far failed.


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